Antibacterial polyurethane materials with silver and copper nanoparticles

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ScienceDirect Materials Today: Proceedings 4 (2017) 87–94

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Functional nanomaterials in Industrial Applications: Academic - Industry Meet

Antibacterial polyurethane materials with silver and copper nanoparticles Yurii Savelyeva, Alexey Gonchara, Boris Movchanb, Alexey Gornostayb, Sergey Vozianovc, Adel Rudenkoc, Rita Rozhnovaa, Tamara Travinskayaa* a

Institute of Macromolecular Chemistry, NAS of Ukraine ,Kharkovskoe Shosse, 48, Kiev 02160, Ukraine b Paton Institute of ElectroWelding, NAS of Ukraine, Bozhenko str., 11, Kiev 03650, Ukraine c Institute of Urology of NAMS of Ukraine, Kotsyubinskogo str., 9A, Kiev 04053, Ukraine

Abstract New biologically active polyurethanes with silver and copper nanoparticles in their composition were prepared by saturation of liquid polyether (the initial component for polyurethane synthesis) with nanoparticles (Ag, Cu), followed by synthesis of polyurethane. The results of these studies allow consideration of these materials as medical suppliers with antibacterial properties. The main problem of their creation is uniform distribution of the metal nanoparticles inside the polymer matrix without changing the physico-chemical properties of the latter. Obtaining of metal nanoparticles’ colloid in a liquid polyoxytetramethylene glycol, MM 1000 was carried out by electron beam evaporation technology and vacuum deposition. This method allowed creating, on the basis of obtained colloids, the metal-containing polyurethane materials with targeted properties and structure that are determined by diisocyanates and chain extenders nature. Biological study has shown that polyurethanes containing Cu and Ag nanoparticles possess bactericidal/bacteriostatic effect against bacteria, fungi and yeast-like fungi. Polymer samples did not exhibit toxicity to the tissue culture cells and there were no biodegradation products observed in the culture medium. Resulting biologically active metal-containing polyurethane materials can be processed into products for medical purposes (implants, drug delivery systems, antimicrobial coatings for biomedical devices and antimicrobial packaging) by standard methods of polyurethane processing, since the presence of metal nanoparticles in their composition does not affect the physical properties of the polymer.

* Corresponding author. Tel.: +38-044-291-03-37; E-mail address: [email protected] 2214-7853 © 2017 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of the Conference Committee Members of Functional Nanomaterials in Industrial Applications.

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© 2017 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of the Conference Committee Members of Functional Nanomaterials in Industrial Applications. Keywords: biologically active polyurethane; copper and silver nanoparticles; bactericidal/bacteriostatic properties

1. Introduction In recent years, due to the development of nanotechnology and supramolecular chemistry a lot of attention is focused on the production of polymer systems containing metal nanoparticles, which exhibit unique physical and chemical properties [1, 2]. These particles have a large reactivity and increased tendency to atomic and ion exchange. When intercalated into polymeric matrix they impart new properties to nanocomposites. Stability of nanoparticles in polymer systems is maintained for a long time. The copper and silver nanoparticles are of particular interest since they exhibit biological and antibacterial activity and can be successfully used in medicine, agriculture and catalysis [3- 6]. Methods for the synthesis of silver nanoparticles may be divided into chemical and physical. Recover of silver particles from its salts aqueous solutions in the presence of various stabilizers is the most widespread chemical method [7, 8]. The hydrogen and hydrogen-containing compounds (tetrahydroborates and alkali metal’ citrates, hypophosphites, alcohols, metalorganic compounds) are used as reducing agents [9]. Reduction of silver nanoparticles can occur both on the surface of pre-synthesized carriers in the presence of a reducing agent, and on the stage of monomers polymerization. The physical methods include spraying or mechanical grinding of bulk material [10, 11]. Among the physical methods used for the formation of nanoparticles of the metal-liquid system the method of ionic synthesis is the commonly used one. A method that allows to fulfill a controlled synthesis of metal nanoparticles (MN) at different depths below the surface of the irradiated matrix is described in paper [12]. The method of pulsed laser ablation of metal described in [13] allows to obtain MN directly in the fluid. Magnetron sputtering method presented in [14] has significantly expanded the scope of the nanoparticles production. This method also allows to obtain MN directly in liquid, spraying the target beyond the liquid matrix bulk. Among other methods of obtaining silver nanoparticles a photoreduction of silver salts and electrochemical methods should be noted [15, 16]. These methods allow to obtain nanoparticles to study the processes of their formation and growth, however, such methods are not suitable for producing nanoparticles on an industrial scale. Method of vapor stream deposition, which refers to physical methods of metal nanoparticles producing, enables the production of high performance colloidal solutions with different concentrations of nanoparticles of various metals (silver, copper) up to 1000 ppm. The advantages are the follows:  control of the evaporation rate and density of the vapour stream  control of the amount of evaporated substance  method eliminates the need for stabilizers and reducing agents  release from foreign impurities during producing of metal nanoparticles and thus, avoid of the need for further purification of the particles Meanwhile, in the majority of the fields of nanoparticles’ application, such as microelectronics, medicine, spectroscopy, catalysis, the presence of even the smallest impurities is unacceptable [17]. It should be noted that all the processes are carried out in a vacuum and do not lead to possible damage of the polymer structure, ensuring its purity. Polyurethanes are known as biocompatible polymers having valuable properties such as strength, flexibility, elasticity [18]. Due to ability of wide variation of properties they are used in virtually all fields of human activity. One of the basic components in polyurethanes synthesis is polyester/polyether. Both the polyester and polyether may be used depending on required properties of polyurethane material: strength, elasticity, crystallinity, glass transition temperature and melting point. The purpose of this paper is to obtain the biologically active polyurethane materials containing silver and copper nanoparticles and study their antibacterial activity.

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2. Experimental 2.1 Materials Polyether polyoxytetramethylene glycol POTMG 1000 [Aldrich], 4, 4'-diphenylmethane diisocyanate (MDI) [Aldrich] as diisocyanate constituent and 1,4-butanediol (BD) [Aldrich] as chain extender were used for the synthesis of metal-containing polyurethane materials. Selection of POTMG-1000 as the polyether to create biologically active polyurethane materials has been specified by necessity of obtaining flexible and durable material for medical application. Furthermore the melting point of this polyether is 24 – 26 oC that allows to condense the metal vapors in liquid polyether under normal conditions. 2.2 Methods 2.2.1. Preparation of colloidal solutions of metals in POTMG -1000 Preparation of colloidal solutions of metals in POTMG -1000 was carried out in a laboratory unit UE-142. The deposition of the vapor flow from the vapor phase and the unit scheme is in detail described in the [19]. 2.2.2 Determination of metal content in the colloid composition Determination of metal content in the colloid POTMG 1000 - Ag, Cu was carried out by atomic absorption analysis [20] on the atomic absorption spectrophotometer with flame atomization AAS-1N (Carl Zeiss Jena) 2.2.3 Study of colloids by laser correlation microscopy (LCS) The distribution range of the formed metal nanoparticles was determined by LCS on the instrument "Zeta Sizer-3" (Malvern, Great Britain) 2.2.4 Study of colloids by transmission electron microscopy (ТEМ) The method of TEM in transmission mode was used for study of sediment after separation of the liquid POTMG 1000 matrix from the POTMG – Ag/Cu colloid. TEM micrographs were processed using specialized software complex computer image analysis «Media cybernetics image analysis program» Image-Pro Plus version 6.0 with following statistical analysis. Obtained data reflect the 95% confidence interval of average particle size values. The mathematical processing of the results was performed using the methods of variation statistics by means of statistical analysis software Microsoft Excel. 2.2.5 Synthesis of metal-comprising polyurethane nanomaterials based on Ag-POTMG and Cu – POTMG colloids Synthesis of Ag-POTMG and Cu-POTMG colloids’ based metal-comprising polyurethane nanomaterials includes a process of obtaining of linear polyurethane based on POTMG- 1000, 4, 4'-MDI and butanediol (BD). The synthesis includes two stages: synthesis of MDI and extension of macromolecule by polycondensation reaction of MDI and BD. Elongation process takes place in an aprotic polar solvent dimethylformamide (DMF), [Aldrich]. The presence of metal nanoparticles does not affect the chemical reactions at all stages of the synthesis, which allows to obtain a metal comprising polyurethane of given structure. This method is versatile: the use of POTMG-1000 metal colloids for the following synthesis of metal comprising polyurethanes of different structures and properties [21]. 2.2.6 Tensile tests of polyurethane materials Tensile tests of polymer systems were performed on a tensile machine FU-1000 (Germany) at a tensile rate of 100 mm / min and 25 C.

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2.2.7 Toxicological studies of polyurethane materials Toxicological studies were carried out on tissue culture of subcutaneous fat of wistar rats [22]. The method of tissue culture, which is a model test system in toxicological experiment was used for the toxicological studies As a source of cells the subcutaneous tissue of white laboratory rats was used; it caused the fibroblasts growth under the cultivation conditions. Cultures were investigated by explantation into the plasma clot in Carrel bottles. On the 3, 5, 7, 10 and 14 day of tissue cultivation the model medium 199 in the experimental group was replaced by extracts from test samples. Extract was prepared in a 1: 1 ration of the area of sample surfaces (cm 2) to a volume of model medium (cm3). Cultured tissues were served as a control. In order to standardize the nature of the cultures growth their growth zones were classified on compact, networklike, and a zone of migratory cells. The character of location of the growing cells was taken as the criterion for the selection of these zones. The zone of dense arrangement of growing cells was attributed to the compact growth zone. The zone of location of anastomosing and branched cellular bands was referred to the network-like. The zone of migratory cells was determined according to the tops of cellular bands and cells laying separately that had grown into solid phase of nutrient medium [23-24]. 2.2.8 Study of the biological activity of polyurethane materials The biological activity of metal comprising polyurethane materials was studied in relation to a number of pathogenic microorganisms: Bacteria S.aureus, E.aerogenes, P.mirabilis, E.coli, K.pneumoniae, P.aeruginosa; Yeast-like fungi Candida albicans and Candida non-albicans; Mould fungi (micromycetes) Aspergillus flavus, Aspergillus niger, Alternaria alternate, Penicillium spp., Paecilomyces lilacinus. The following nutrient solutions were used: blood agar, nutrient agar (for bacteria), agar Saburo - for candida and micromycetes. The nutrient solutions (20 mL of each) were overflowed per Petri dish of 90 mm diameter. The suspension of microorganisms was prepared in saline (0,95% NaCl) using the instrument DENSI LA METR II and it was adjusted to a concentration of 1x106 CFU / ml. Candida species were used in a concentration 1x103 CFU/ml. Micromycetes – 1x104 CFU / ml [25]. During the time of experiment preparation, the test tubes containing the suspension of bacteria and fungi were placed in a shaker. Next, 1 ml of each sample was transferred to the agar surface and with the Drigalski spatula was uniformly distributed on the surface. An excess of suspension was removed with disposable pipette. The dishes were left near the burner for 30 minutes for drying. Then the polyurethane discs, comprising the stated concentrations of nanometals were applied to the surface and placed to a thermostat at 37 oC for bacteria and at 28 oC for micromycetes. Primary results were taken at 24 hours (for bacteria), 48 hours (for Candida fungi) and 10 days (for micromycetes). To eliminate the secondary growth of pathogens, all dishes after the growth fixation were kept for another month. Then a second record of results was performed. After the primary and in one month registration the scrapings from the surface of each polyurethane disc containing various concentrations of nanometals were made, and inoculations on appropriate media were carried out. 3. Results and discussion 3.1 Results of (ТEМ) of POTMG-1000/metal nanoparticles colloid solutions TEM micrographs of colloid solutions of metals in POTMG-1000 (Fig. 1) on silver example display that metal particles in POTMG-1000 matrix mainly have a spherical shape. According to the TEM data the histograms of Ag nanoparticles’ distribution in POTMG-1000 volume were built (Fig. 2), which represent the dependence of the particle size of Ag (nm) on their ratio in POTMG-1000 (%) volume.

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Ratio in the volume, % Ñîîòíîøåíèå â îáúåìå, %

40

30

20

10

0 0

Figure 1 – Micriphotographs of Ag-POTMG-1000 colloid

30 60 90 Ag nanoparticle size, nm. Ðàçìåð ÷àñòèö, íì

120

Figure 2– Histogram of Ag nanoparticles distribution in POTMG-1000 volume

Figure 3 – Nanoparticle size distribution of Ag-POTMG by LCS

According to histogram’s data the Ag particles are distributed in the size range of 10 - 110 nm, an average particle size amounts to 58 nm. A fraction ranged in 30 - 90 nm amounts to 86% from the total particles quantity. The area occupied by Ag particles amounts to 42% of the total area. LCS Results (Fig. 3) shows the presence of particles in the range of 10 - 140 nm. The number of particles is 100%; weight is also 100%. The most probable particle size is 52.2 nm. The average polydispersity is 1 (unit). As the results of the synthesis a polyurethane solutions with metal nanoparticles in an organic solvent DMF have been obtained. After gradual evaporation of the solvent the samples of polyurethane metal comprising film materials have been formed. The composition and properties of the obtained polyurethane materials are presented in Table 1. Table 1 Composition and properties of biologically active polyurethane materials Composition and Cu:Ag Cu Cu Cu properties [200:69] [666,7] [308] [200] Ag, ppm 69 Cu, ppm 200 666,7 308 200 Melting point, Тm.С 136 – 138 Degradation 175 – 176 temperatureТd.С Tensile strength, МPа

32 – 35

Ag [98] 98 -

Ag [27] 27 -

Ag [15] 15 -

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Nanoparticles of silver and copper do not affect the thermoplastic nature and strength of the polyurethane nanocomposite material, which allows to process the obtained polyurethane material by extrusion. 3.2 Biological activity Results of mycological and microbiological study of polyurethane metal comprising nanomaterials show that bacteria and fungi of the genus Candida were highly sensitive to the action of all the studied concentrations of copper and combinations Cu (200) and Ag (68,745 ppm). It should be noted that growth inhibition zone of bacteria and Candida around the disk was not observed; however, the inoculation of scraping/smear from the surface of the disk was sterile as at the primary registration, and in a month. The results of mycological and microbiological study are summarized in Table 2. Table 2 –Biological study of metal comprising polyurethane nanomaterials Strains studied Polyurethanes with Cu, Ag and Cu+Ag, ppm Cu:Ag [200:69]

Cu [666,7]

Cu [200]

Ag [98]

Ag [27]

Ag [15]

0/0

Cu [308] Bacteria 0/0

S.aureus

0/0

0/0

0/0

0/0

0/0

E.coli K.pneumoniae

0/0 0/0

0/0 0/0

0/0 0/0

0/0 0/0

0/0 0/0

0/0 0/0

0/0 0/0

P. mirabilis

0/0

0/0

0/0

0/0

0/0

0/0

0/0

E. aerogenes P. aeruginosa

0/0 0/0

0/0 0/0

0/0 0/0

0/0 0/0

0/0 0/0

0/0 0/0

С.albicans C.non-albicans

0/0 0/0

0/0 0/0

0/0 0/0

0/0 0/0

0/0 0/0

0/0 0/0

Aspergillus flavus A.niger

0/2 0/0

0/2 0/0

0/3 0/0

0/4 0/2

0/4 0/2

0/4 0/2

0/3 0/2

Alternaria alternate

0/0

0/0

0/3

0/3

0/3

0/3

0/3

Penicillium spp.

0/0

0/0

0/3

0/3

0/3

0/3

0/3

0/#

0/4

0/0 0/0 Candidas 0/0 0/0 Micromycetes

Paecilomyces lilacinus 0/3 0/3 0/4 0/4 0/# # - continuous growth 0 – no inhibition zone around the disk n/d : numerator – primary registration, denominator – secondary growth of the test culture on the disk, mm

The results of studying of the effect of different nanometals’ concentrations in polymer on micromycetes have shown that in 10 days from the start of the experiment, the surface of the disc remained sterile, but in a month a secondary growth of fungi on the contour of the disk was observed (Table 2). At that the disks with Cu concentration (666,7 ppm and 308 ppm), and combined Cu and Ag content (Cu - 200 ppm; Ag - 69 ppm) were the most active against micromycetes cultures polyresistant to antibiotics. The results revealed that the sensitivity of fungi to copper and silver was strain-dependent. Biological study has shown that polyurethanes containing nanoparticles of Cu and Ag exhibited bactericidal properties in relation to both gram-positive and gram-negative bacteria, and yeast-like fungi. The same polyurethanes demonstrated bactericidal and bacteriostatic effect in relation to fungi - micromycetes. 3.3 Toxicological evaluation of polyurethane Investigation of the growth and development of cellular elements of hypoderm of white rats was carried out for 3, 5, 7, 10, 14 days. The first signs of growth, which were apparent by migration of single elongated cells, as well as by

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single migrating fibroblast elements were spindle-shaped, both in the control and in all test samples and were observed until the end of 3 days. (Fig. 4)

Figure 4 - Beginning of growth in the culture of subcutaneous fat of rats. Control.

Further, the activity of fibroblastic elements’ growth has increased. In 5-7 day in Carrel vials with samples as well as in the controls, the areas of growth were presented by three zones: • a compact, consisting of polygonal and spindle-shaped cells, • reticular composed of bundles and strands cells located reticular and • zone of migratory fibroblastic elements. On the 10th day of tests a compact and reticular zones and zones of migrating cells for all tested samples increased. On the 14th day of the study the cell population entered a phase of degeneration, which was manifested in large vacuolization of the cytoplasm and its granular degeneration in the cells of all studied and control samples, which was typical for this term of the culture (Fig.5).

Figure 5 – Granular cytoplasmic degeneration in 14 days in the control

Thus, it was found that during 14 days of cultivation no biodegradation products were stand out into the culture medium, and the samples did not exhibit toxicity on the cells of tissue culture. Conclusions New biologically active polyurethanes with silver and copper nanoparticles in their composition were prepared by saturation of liquid polyether POTMG – 1000 (the initial component for polyurethane synthesis) with metal nanoparticles (Ag, Cu), followed by synthesis of polyurethane. The electron-beam evaporation and vacuum deposition of inorganic materials technology allowed to obtain the colloidal solutions of Ag and Cu nanoparticles (diameter ranging from 10 to 120 nm) in the POTMG – 1000. Average particle size was 52 nm, the fraction, located

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in the range of 30-90 nm was 86% of the total number of particles. Obtaining of biologically active metal-containing polyurethane materials based on abovementioned technology is a universal method for creation of biologically active materials with targeted properties. Рolyurethanes containing Cu and Ag nanoparticles exhibit bactericidal properties against both gram-positive and gram-negative bacteria, and yeast-like fungi. It was shown that studied nanostructured Cu/Ag polyurethanes possess a strain-dependent fungicidal effect against A.niger, Alternaria alternate and Penicillium spp. In other studied cases they possess the fungistatic effect. It was found that during 14 days of polymer samples’ cultivation the tested samples did not exhibit toxicity to the cells of the tissue culture and no any products of biodegradation were observed in the culture medium. Resulting biologically active metal-containing polyurethane materials can be processed into products for medical purposes (implants, drug delivery systems, antimicrobial coatings and packaging, catheters, drains, film, mesh and so on) by standard methods of polyurethane processing, since the presence of metal nanoparticles in their structure does not affect the physical properties of the polymer. References 1. A.D. Pomogailo, А.S. Rosenberg, I.Е. Ufliand, Metal nanoparticles in polymers. М.:Chemistry, (2000) 672. 2. A.D. Pomogailo, V.N. Kestelman in: Metallopolymer nanocomposites; Springer-Verlag Berlin Heidelberg (2005), Vol. 81, pp. 469-576. 3. P. Jain, T. Pradeep, Biotechnology and bioengineering. 90 (2005), 59 – 63. 4. Maribel G. Guzmán, Jean Dille, Stephan Godet. Int J Chem&Biol Eng. (2009) 104-111. 5. Lamabam Sophiya Devi and S. R. Joshi. Mycobiology. 40 (2012) 27–34. 6. Yu.V. Savelyev, О.N. Gonchar, B.О. Movchan, О.V. Gornostay, А.V. Rudenko. Ukrainian Patent 94092 , October 27, 2014. 7. C. Song, Y. Lin, Z. Hu. Nanotecnology. 15 (2004) 962 – 965. 8. Chou, K-S., Huang K-C., Lee H-H. Nanotecnology 16 (2005) 779 – 784. 9. Shekhar Agnihotri, Soumyo Mukherji and Suparna Mukherji. The Royal Society of Chemistry. 4 (2014) 3974-3983. 10. T. Oates, A. Mucklich, Nanotecnology. 16 (2005) 2606 – 2611. 11. J. Lu, K-S. Moon, J. Xu, C. P. Wong, J. Mater. Chem, 16 (2006) 1543 – 1548. 12. A. L. Stepanov, R. I. Khaibullin, V. F. Valeev, Yu. N. Osin, V. I. Nuzhdin, I. A. Faizrakhmanov, Technical Physics. 54 (2009) 11621167. 13. P. Wagener, S. Barcikowski. Laser Technology. (2011), 20-22. 14. Zhenhua Tang, Ying Xiong, Minghua Tang, Yongguang Xiao, Wei Zhang, Meiling Yuan, Jun Ouyang and Yichun Zhou. J. Mater. Chem. C. 2 (2014) 1427-1435. 15. K. A. Bogle, S. D. Dhole, V. N. Bhoraskar. Nanotecnology. 17 (2005) 3204 – 3208. 16. P. K. Sudeep, P. V. Kamat. Chem. Mater. 17 (2005) 5404 – 5410. 17. Vincenzo Amendola, Stefano Polizzi and Moreno Meneghetti. Langmuir. 23 (2007) 6766-6770. 18. Agosta M., Polyurethane Technology. Coatings World, June 2002, p. 36. 19. O.V. Gornostay, I. S. Kovinsky. Electrometallurgy Today 2(107) (2012) 50-53. 20. I. Khavezov., D. Tsalev. Atomic-absorption analysis. L.: Chemistry. 1983 p.144. 21. Yu.V. Savelyev, A.N. Gonchar, B.О. Movchan, О.V. Gornostay, С.О. Vozianov, А.V. Rudenko. Ukrainian Patent 98460, April 27, 2015. 22. Toxicological-hygienic and preclinical trials of polymer materials and based products of medical purpose. Methodical instructions, Naukova Dumka, Кiev, 2009. p 97. 23. V.P. Yacenko, N.А. Galatenko, G.А. Pkhakadze. Cytology and Genetics 4 (1984) 280–284. 24. N.А. Galatenko, V.P. Yacenko, G.А. Pkhakadze. Doklady Academii Nauk Uktainy. 9 (1982) 54–58. 25. Standard 9.048...9.053-75 (91). Materials and products. Test methods for microbiological stability.